Dataset Information

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR.

ABSTRACT: The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

SUBMITTER: Zhao X

PROVIDER: S-EPMC9735513 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR.

Zhao Xiaoli X He Chengyong C Gao Dehang D Xu Tengfei T Li Xiaofeng X Liu Junjie J Li Shifang S Wang Hongqing H

Plants (Basel, Switzerland) 20221205 23

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open r ...[more]

PMID: 36501425

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Project description:Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Dataset Information

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR.

Publications

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR.

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